This invention relates generally to the provision of a single instrument wherein an electronic particle study device of the Coulter type operating upon the Coulter principle is combined with apparatus for photometric measurement of hemoglobin content of a single sample of blood.
As such, the instrument is an improvement upon the instrument described in U.S. Pat. No. 3,743,424 and as well, provides hemoglobin measuring capabilities which comprise an improvement over U.S. Pat. Nos. 3,927,317 and 3,622,795.
The measurement of the hemoglobin content of blood involves the preparation of a suspension of blood cells in a saline electrolyte solution. The sample is lysed, that is, an agent is added which breaks down the red blood cells to release their content into the solution. This solution and the resulting suspension contain the coloring matter of the red blood cells and the unaffected white blood cells.
The operation of the counting device portion of the invention is disclosed in U.S. Pat. Nos. 2,656,508 and 2,869,078. The first of these describes the general principle which has been referred to above as the Coulter principle, that is, causing particles to pass through a minute aperture whose effective impedance is changed with the passage of each particle in an amount which is proportional to the size of the particle.
The apparatus which utilizes the Coulter principle directs an electric current across a scanning aperture, and detects and counts the signals produced by the particles of a known volume as they pass through the aperture. The second of these patents describes apparatus which automatically starts the operation of the counting device merely by turning a stopcock, meters the required volume to pass through the aperture, and then turns the counting device off.
The apparatus disclosed in U.S. Pat. No. 3,743,424 included an electronic particle counting device operating on the Coulter principle which was combined with an optical hemoglobinometer such that at some step in the sequence of events of the operation of the counting device, the operation of the optical hemoglobinometer is started. The same sample suspension that is utilized in the counting device is utilized also in the hemoglobinometer by means of a thief or fluid connection from a vessel of the counting device to the hemoglobinometer. The action of the counting device in starting the hemoglobinometer is automatic.
The hemoglobinometer of said U.S. Pat. No. 3,743,424 involved the use of a standard solution which was viewed by an optical colorimeter to obtain a value of the absorbance of the standard solution. This information was stored in a suitable storage circuit and the test sample or unknown was viewed by the same colorimeter and its absorbance value compared with that of the standard solution. Thus a supply of standard solution was necessary and a quantity required to be introduced to the area traversed by the light beam of the colorimeter every time a determination was to be made upon a test sample. A manometer/syphon apparatus was utilized to move the sample suspension both through the aperture of the counting apparatus and through the hemoglobinometer. Limitations as to the quantity of standard solution available pose a significant problem to widespread use of the aforementioned combination notwithstanding its capability to provide both white cell count (WBC) and hemoglobin content (HGB).
U.S. Pat. No. 3,566,133 teaches a voltage storage and rundown circuitry for use in photometric analysis and U.S. Pat. No. 3,622,795 teaches a flow-through system for measuring the absorbance of a sequence of test samples as related to a reference liquid which is introduced sequentially alternately with the test samples, as in the combined counter and hemoglobinometer of U.S. Pat. No. 3,743,424.
According to U.S. Pat. No. 3,566,133, just prior to the introduction and measurement of each sample, a quantity of the reference liquid is fed to the photometric apparatus and its transmittance measured to provide a voltage representative thereof. Like the combined counter/hemoglobinometer, no provision was made for storing a "reference" value internally so that the use of the standard solution could be eliminated. The U.S. Pat. No. 3,927,317 provides apparatus and electrical circuitry therein which can provide an electrically stored standard value which is that of the voltage value of a blanking solution. However, the blanking solution is not a fixed standard and can vary from batch to batch as to transmittance so as to be practically an unknown itself.
Accordingly, an initial calibration adjustment must be made for each blanking solution. To accomplish such calibration, a calibration control solution is compounded having a known photometric response. Calibration is not an easy or precise operation and is subject to many variations which reduce accuracy. Error is introduced by the circuitry itself due to component aging, power fluctuations, and other common electronic variables experienced with various components and circuit conditions. It is desirable to eliminate such error and increase accuracy and precision in providing hemoglobin content measurements in any proposed combined cell counter and hemoglobinometer.
Any testing of a sequence of liquid samples presents liquid dispensing, handling, measuring and disposal problems. Where the instrument is desired to be of "flow-through" type, the various components including vessels, cuvettes, tubing, etc., require filling, rinsing, draining, refilling, etc., all in timed relationships with respect to volumetric and photometric measurements.
U.S. Pat. No. 3,622,795 teaches a flow-through photometer having a fluid handling system in which an automatically supplied rinse liquid having a known transmittance was employed as the blanking solution. Means were provided to accomplish the measurement of hemoglobin in a lysed blood sample by manual pouring of a sequence of samples into a cuvette or test vessel alternately with automatic dispensing of the blanking solution. These described expedients still were not fully satisfactory.
In U.S. Pat. No. 3,566,133 the transmittance of the reference standard was electrically stored momentarily as a voltage applied to a storage capacitor and then discharged down to a voltage value having a predetermined value representative of a sample against which the reference is being compared. In such apparatus, the time duration of the voltage rundown was calibrated to provide a readout of the absorbance of the sample, for example. However, while this circuit was a distinct improvement over the prior art, plural introductions of the calibration solution were required to accomplish calibration of the instrument. In many apparatus where a reference value was stored, the value generally was lost when the readout was made and hence repeated full runs had to be made with a calibration solution in order to complete the calibration process. This was time consuming as well as expensive.
U.S. Pat. No. 3,927,317 provided electric circuitry for a photometer which was capable of storing the voltage value of a blanking solution for long periods of time. The blanking solution is employed initially with a calibration control solution for calibration purposes and then is employed for comparative voltage rundown measurements of a sequence of samples. Thus for calibration, the transmittance of the blanking solution is stored as a voltage on a capacitor which is permitted to discharge until equal to the voltage representative of the transmittance of a calibration solution of known or assayed HGB.
It would be highly desirable to combine with the improved hemoglobin measuring apparatus of the type disclosed in U.S. Pat. No. 3,927,317 a particle counting device of the Coulter type such as employs the counting system disclosed in U.S. Pat. No. 3,733,548. A considerable deterrent to such combination is the required use of vacuum to draw liquid suspension through the scanning aperture during the counting of particles. With the combination considered, vacuum is required generally only when necessary to move fluid through the scanning aperture. This is only a part of the proposed operating cycle. However, the vacuum systems available all require continuously generated vacuum pumps, attendant vacuum bottles, and supporting hardware. Danger of spillage and breakage accordingly are presented with such systems last described. Space utilization problems also occur which must be solved before an acceptable combined instrument can be provided which is a marketable product.
Other problems involved in the combined instrument result in operator or support personnel errors occasioned during the operation or in preparation of the necessary sample solutions. The various functional devices should be monitored with appropriate warnings provided for alerting the operator to mishaps which may occur when they occur.
Another problem which is encountered in the handling of successive samples particularly in a flow-through device is the danger of carryover of one sample to the next and/or, if the sample is emptied and replaced by rinse liquid, for example, dilution of the next sample by residual rinse liquid being left behind when the rinse liquid is drained from the test vessel, or by bubbles, etc., remaining within the drained test vessel. Carryover reduces precision and increased precision is a goal for any new instrument. While rinsing a test vessel subsequent to release of a sample therefrom is helpful in reducing carryover of one sample to the next to appear sample, there remains the problem of compensating for the possible dilution of the sample by the residual rinse liquid. It would be highly desirable materially to reduce carryover effect to the point that it will play no, or at least a negligible, part in preventing achievement of highly improved precision during operation of the combined instrument.